Turnover of 125I-labelled tissue kallikrein following intraduodenal or intravenous administration.

UNLABELLED: Tissue kallikrein is released in the body both physiologically and in many inflammatory disorders. Little is, however, known about the turnover of released tissue kallikrein in humans. Approximately 1 mg of tissue kallikrein (mol wt 43,000 Da) was purified from 85 L human urine by: (1) ultracentrifugation, (2) filtration through an aprotinin-coupled Sepharose 4B column, followed by (3) gel filtration over a Sephadex G-75 column. The elimination, after intraduodenal or intravenous administration of purified tissue kallikrein radiolabelled with 125I, was followed by collecting serial samples of plasma, urine and faeces from three volunteers. Within 72 h, about 96% of the intraduodenally administered radioactivity had been excreted in urine, and approximately 5.4% in faeces, mainly as 125I. No intact 125I-tissue kallikrein was found in plasma, urine or faeces after the intraduodenal instillation of the protein. The plasma half-life of 125I-tissue kallikrein up to 3 h after intravenous injection was 9 min and, thereafter, 20 h. The 125I-tissue kallikrein was quickly bound to a plasma protein with a mol wt of about 67 kDa, but some of the radioiodinated tissue kallikrein was still unbound 15 min after injection, judged by gel filtration on Sephadex G-200 columns. Most of the radioactivity was excreted in the urine as 125I, but about 4-6% was recovered as free 125I-tissue kallikrein. CONCLUSION: The use of tissue kallikrein as an oral drug appears, therefore, to be useless. Tissue kallikrein released into plasma seems to be quickly bound to a protein with a mol wt of 67 kDa, probably kallistatin or Protein C inhibitor, but some tissue kallikrein seems to be unbound and may have some physiological or pathophysiological action. The unbound tissue kallikrein is, at least partly, cleared from the circulation by the kidneys, and tissue kallikrein in the urine may partly be derived from plasma.

Tissue kallikrein in severe acute pancreatitis in patients treated with high-dose intraperitoneal aprotinin.

The activation of the kallikrein-kinin system is thought to be one of the pathophysiologic mechanisms in acute pancreatitis. A radioimmunoassay for human urinary tissue kallikrein was developed and used to measure tissue kallikrein peritoneal exudate and plasma from 48 patients with severe acute pancreatitis. All patients were treated with intraperitoneal lavage. One group (n = 22) received high doses of the protease inhibitor aprotinin (aprotinin group), and the other group, saline (control group). Levels of kallistatin in peritoneal exudates and plasma were measured with an enzyme immunoassay. A large increase in tissue kallikrein was observed in the peritoneal exudate, which declined in both groups after multiple lavages. Complexing of liberated tissue kallikrein with kallistatin was evidenced by gel filtration in both peritoneal exudates and plasma in both groups. The decrease in kallistatin observed in both peritoneal exudate and plasma is therefore regarded as being due not only to repeated lavage, but also to true consumption of the binding protein. Some of the liberated tissue kallikrein in the peritoneal fluid and plasma was complexed to aprotinin. In the control group, six patients were operated on because of pancreatic necrosis, compared with none in the aprotinin group. The levels of tissue kallikrein in the lavage fluid were lower in the control group, but this was the result of the very low tissue kallikrein values in the six patients operated on for pancreatic necrosis. Levels of kallistatin in plasma and peritoneal exudate in these six patients were lower on the day of admission compared with the other patients, and the plasma levels continued to be lower during the first week. Large amounts of tissue kallikrein were found to be released into the peritoneal exudates in acute pancreatitis. Lavages effectively cleared the released tissue kallikrein. The tissue kallikrein was complexed to kallistatin, whereas in the aprotinin group, also to aprotinin both in plasma and in peritoneal fluid. The partitioning of kallikrein between the two inhibitors was the result of the interaction between enzyme and inhibitors and the turnover of the complexes formed. The low admission levels of kallistatin in the six patients operated on because of pancreatic necrosis suggest that kallistatin may act as an early marker of severity in acute pancreatitis.

Interactions in vitro and in vivo between porcine tissue kallikrein and porcine plasma proteinase inhibitors.

The elimination of radio-iodinated porcine tissue kallikrein, after intravenous injection in the pig, showed a rapid initial clearance from plasma (T1/2 approximately 10 min), followed by a phase of slower elimination (T1/2 approximately 100 min). Gel filtration of plasma samples showed complexes with alpha 1-alpha 2-macroglobulin (A1a2-M) and alpha 1-proteinase inhibitor (A1-PI), which decreased with time. The urinary excretion of undegraded tissue kallikrein was about 1.8%. Gel filtration of urine showed a minor peak representing free tissue kallikrein and a major peak representing degradation products. On average, 8.3% was found in the liver and 1.3% in the kidneys post mortem, indicating that these are the primary organs for the elimination of tissue kallikrein. The in vivo findings were supported by in vitro experiments. A1a2-M were found to be the major inhibitors of tissue kallikrein, when a mixture of the enzyme and porcine plasma was analysed by gel filtration, immunoelectrophoresis, crossed immunoelectrophoresis and autoradiography. A1-PI was only a minor inhibitor of tissue kallikrein. Both the A1a2-M and A1-PI complex formation was found to be time-dependent and slow; unbound glandular kallikrein was still detected after 12 h, even when there was a molar surplus of A1a2-M and A1-PI. The complexes with A1a2-M and the unbound tissue kallikrein were found to be enzymatically active against low-molecular-weight chromogenic substrate. The total tissue kallikrein-inhibiting capacity of plasma seemed to be exceeded at a concentration of 800 K U/l when analysed using the rat uterus bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the release of tissue kallikrein in experimental pancreatitis in the pig.

The activation of the kallikrein-kinin system is thought to be one of the pathophysiological factors in acute pancreatitis. A radioimmunoassay for porcine, pancreatic tissue kallikrein was developed and used to measure levels in normal plasma and peritoneal fluid and in experimental, bile-induced (group A) and bile trypsin-induced (group B) acute pancreatitis in the pig. Normal porcine plasma and peritoneal fluid contained about 2.17 +/- 0.11 and 1.91 +/- 0.19 microgram/l (SEM) tissue kallikrein, respectively. In experimental, acute pancreatitis there was a rapid rise in the plasma level of tissue kallikrein, followed by a slow increase to a final value of about 150% of the normal plasma level in both groups. In the peritoneal exudate a large increase (200-fold in group A and 2,000-fold in group B) in tissue kallikrein was seen, with a maximum within about 1/3 of the survival time, followed by a slow decrease until death in group B. In group A a smaller second peak was seen at about 2/3 of the survival time. Gelfiltration of peritoneal exudates showed complexes with alpha 1-, alpha 2-macroglobulin (alpha 1 alpha 2-M), and alpha 1-proteinase inhibitor (alpha 1-PI) and a large portion of free tissue kallikrein. The complexes with alpha 1 alpha 2-M and the free tissue kallikrein were found to be enzymatically active when tested on chromogenic tripeptide substrate. The presence of large amounts of free and active tissue kallikrein in the peritoneal exudate leads us to the conclusion that tissue kallikrein may be a major cause of local release of kinins in acute pancreatitis.